Transcriptional control is a major determinant of gene expression in higher eukaryotes. DNA viruses provide valuable models for studying the transcriptional activation of eukaryotic cellular and viral genes. The long-term objective of this proposal is to develop a detailed understanding of the molecular mechanisms by which viral trans-acting immediate-early proteins activate transcription from well characterized cis-acting control sequences, resulting in regulated gene expression. These problems will be studied both by direct viral infection of cells and the transient expression of transfected genes in cultured cells. Using in vitro mutagenesis techniques, we will introduce mutations into viral immediate-early genes and their ultimate sites of action, viral promoters. The transcriptional activity of these viral mutants will be characterized in vivo. We will attempt to express and isolate large amounts of these mutant and wild type viral immediate-early proteins from bacteria to be used for in vitro studies. In addition, we will attempt to develop a regulated nuclear transcription system to facilitate a more direct analysis of transcriptional activation by viral immediate-early gene products. Through these various approaches, we hope identify the active viral immediate-early protein(s), delineate the cis-acting sequences required for their action, determine with which transcription component(s) these viral proteins interact, and study these interactions to determine the mechanism by which these viral proteins function. Furthermore, we will identify cellular genes that are responsive to trans-acting viral proteins and determine the structural or functional features these genes have in common.